As a molecular switch the ras protein undergoes structural changes that couple recognition sites on the surface of the protein to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 further away from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) pulsed EPR spectroscopy of p21Mn(II) complexes of GMPPNP that probe the link between threonine-35 and the divalent metal ion. In particular, we determine the 17O hyperfine coupling constant of [17Og]Thr 35 is determined to be ~1.1 G in the GTP form. This value is much smaller than reported values (1.6 - 4 G) for 17O-enriched aquo-and phosphato-ligands in other complexes of Mn(II). These results are in agreement with direct, weak coordination of this residue as suggested by Halkides et al. (1994) Biochemistry 33, 4019.